OBJECTIVETo observe epithelial-mesenchymal phenotypes and oxidative stress related protein expressions of the liver cells in a rat model of liver fibrosis induced by bile duct ligation and recanalization.

METHODSTwenty-four male Wistar rats were randomized into 4 groups, including a sham-operated group, two bile duct ligation groups with ligation for 2 and 4 weeks, and a bile duct ligation group with a 2-week ligation followed by a 2-week recanalization. HE staining and Masson staining were used to assess liver fibrosis in the rats, and immunohistochemistry and Western blotting were employed to detect expressions of the epithelial and mesenchymal marker proteins and oxidative stress-related proteins.

RESULTSCompared with the sham-operated group, the rats with bile duct ligation showed obvious liver fibrosis, which worsened as the ligation time extended, accompanied by significantly increased expression of α-SMA, collagen I, NOX(4) and vimetin and reduced E-cadherin expression. Compared with the rats with bile duct ligation for 4 weeks, the rats in bile duct ligation-recanalization group showed obviously lessened liver fibrosis, significantly lowered expressions of NOX(4) and mesenchymal cell maker proteins, and enhanced expressions of epithelial cell marker proteins.

CONCLUSIONBile duct ligation up-regulates mesenchymal phenotype-related proteins and NOX(4) protein expression and down-regulates the expression of epithelial phenotype-related proteins, and these changes can be reversed by subsequent bile duct recanalization.

Actins ; metabolism ; Animals ; Bile Ducts ; surgery ; Cadherins ; metabolism ; Collagen Type I ; metabolism ; Disease Models, Animal ; Epithelial Cells ; cytology ; metabolism ; Hepatocytes ; cytology ; metabolism ; Ligation ; Liver Cirrhosis ; metabolism ; Male ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Phenotype ; Rats ; Rats, Wistar ; Vimentin ; metabolism

Objective To explore the clinical significance of the relationship between the immune function and the pathogenesis of aplastic anemia in children with aplastic anemia(AA),along with the incidence of graft versus host disease (GVHD)by monitoring the changes of T lymphocyte subsets dynamically in +1 ,+3,+6,+1 2 months for blood disease patients after allogeneic hematopoietic stem cell transplantation.Methods Twelve AA patients re-ceived allogeneic hematopoietic stem cell transplantation in Department of Hematology,the Affiliated General Hospital of Beijing Military Region of Anhui Medical University,from January 201 3 to January 201 4,including 4 male and 8 fe-male,with average age of 7.92 years old(3 -1 4 years old)with 5 cases of human leukocyte antigen(HLA)matched and 7 cases of HLA mismatched.The level of T lymphocyte subsets including CD3 +,CD4 +,CD8 +,CD4 +/CD8 +, CD56 +,CD4 +CD25 high +FOXP3 +were monitored with flow cytometry before transplantation and in +1 ,+3,+6,+1 2 months after transplantation dynamically in the peripheral blood.While in the same period the level of T lymphocyte subsets was monitored in 1 2 cases of healthy children at the same period as the healthy control group.Results Fol-lowed up to March 201 5,1 0 cases had abnormal cellular immunity (CD4 +/CD8 + ratio inversion)in the 1 2 AA pa-tients.Compared with the control group,in the AA group,CD3 + was slightly higher,(66.79 ±7.35)% and (62.74 ± 5.58)% respectively(P =0.043),CD4 + was decreased by (33.73 ±7.26)% and (39.54 ±3.46)% respectively (P =0.037),CD8 + was increased by (35.69 ±6.78)% and (25.34 ±4.36)%,respectively (P =0.000),CD4 +/CD8 + decreased by 1 .23 ±0.56 and 1 .78 ±0.34 respectively(P =0.001 )and CD56 + was decreased by (7.46 ± 2.80)% and (1 6.73 ±3.70)% respectively(P =0.000),CD4 +CD25 high +FOXP3 + was decreased by (3.3 ± 1 .5)% and (8.1 ±1 .3)% respectively (P =0.003),whose difference was statistically significant (P <0.05).The lever of CD3 +,CD4 +,CD8 +,CD4 +/CD8 +,CD56 +,CD4 +CD25 high +FOXP3 + had a different degree of recovery after transplantation for all cases and returned to normal in +1 2 months basically.In +1 ,+3,+6,+1 2 months after transplantation,the levels of CD4 +CD25 high +FOXP3 + in GVHD positive group and negative group were (0.4 ± 0.6)% and (1 .6 ±0.7)% respectively,(0.7 ±0.3)% and (2.7 ±0.4)% respectively,(1 .1 ±0.5 )% and (2.9 ±0.7)% respectively,(1 .4 ±0.3)% and (3.6 ±0.2)% respectively,which had statistical significance (P <0.05).Conclusions There was abnormal cell immune function in some cases with AA.After transplantation,the level of CD4 +CD25 high +FOXP3 + is closely related to the acute GVHD,which can be used to predict the occurrence of GVHD.

BACKGROUND:Al ogeneic hematopoietic stem cel transplantation is currently recognized as the first-line therapy for severe aplastic anemia. However, with the popularity of the one-child families, the source of ful y matched hematopoietic stem cel transplantation is limited, so haploidentical hematopoietic stem cel transplantation is favored. OBJECTIVE:To retrospectively compare and analyze the clinical efficacy and safety of haploidentical al ogeneic hematopoietic stem cel transplantation and ful y matched hematopoietic stem cel transplantation for the treatment of severe aplastic anemia. METHODS:Clinical data of 15 patients with severe aplastic anemia (treatment group) who underwent haploidentical al ogeneic hematopoietic stem cel transplantation in the Department of Hematology General Hospital of Beijing Military Region from January 2013 to January 2015 were retrospectively analyzed. Pretreatment regimen was cyclophosphamide, fludarabine, Busulfex, combined with anti-human lymphocyte immune globulin. Donors received granulocyte colony-stimulating factor, and the transplantation method was bone marrow mobilization combined with peripheral blood stem cel transplantation. Combined immunosuppressive agents including cyclosporine A, methotrexate, tacrolimus, were adopted for prevention of graft versus host disease. Another 15 cases of severe aplastic anemia undergoing ful y matched hematopoietic stem cel transplantation served as control group over the same period. Complications and survival of the two groups were statistical y analyzed. RESULTS AND CONCLUSION:By the end of July 2015, the median fol ow-up time of the treatment group was 20.7 months (6-30 months), and hematopoietic reconstruction was achieved in al cases, including four cases of graft versus host disease, five cases of pulmonary infection, three cases of sepsis, and one case died of pulmonary infection, one cases died of sepsis, and two cases died of graft versus host disease. In the control group, the median fol ow-up time was 19.7 months (5-28 months), hematopoietic reconstruction was achieved in al cases. There were three cases of graft versus host disease, four cases of pulmonary infection, one case died of pulmonary infection, and two cases died of graft versus host disease. The total survival rates of the two groups were 73%and 80%respectively, with no significant difference (P=0.67). The haploidentical al ogeneic hematopoietic stem cel transplantation for severe aplastic anemia is safe and effective, and the clinical efficacy is comparable to the ful y matched hematopoietic stem cel transplantation.

OBJECTIVETo investigate the dynamic changes of interlukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in gingival crevicular fluid (GCF) during orthodontic tooth movement, and to discuss the biological significance.

METHODSFifteen patients were chosen as subjects. For each patient, upper and lower canines at one side having one treatment for distal movement by elastic chain served as the experimental teeth, whereas the contralateral ones were used as controls. The GCF were taken before activation and at 1, 24, 48, 72, 168 hours respectively after initiation of the experiment. The levels of IL-1beta and TNF-alpha in GCF were determined by radioimmunoassay.

RESULTSThe levels of IL-1beta and TNF-alpha in experimental group began to increase at 24 hours and reached to its peak value at 72 hours after initiation of the experiment, but their levels returned to baseline at 168 hours. Both of them, however, remained at the baseline level in control group. The changes of the two cytokines level were found statistically significant at 48 and 72 hours (P<0.05) between experimental and control group. No statistically significant were observed before activation and at 1, 168 hours after application of orthodontic forces (P>0.05) between experimental and control group.

CONCLUSIONThe levels of IL-1beta and TNF-alpha in gingival crevicular fluid experience dynamic changes during the early phase of orthodontic treatment, indicate that they might play an important role in the process of alveolar regeneration and tooth movement.

Female ; Gingival Crevicular Fluid ; Humans ; Interleukin-1beta ; Male ; Tooth Movement Techniques ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the changes in cytokines (IL-1beta, IL-2, TNF-alpha) of peripheral blood and cervical mucous of infertile women with mycoplasma infection and the effect of intervention of traditional Chinese medicines (TCMs).

METHODAccording to the results of culture of mycoplasma from genital tracts, 72 patients with positive mycoplasma were randomly divided into the TCM group (38 cases) and the western medicine group (34 cases). The western medicine group was treated with 0.5 g azithromycin for 3 days and consecutively treated for six courses of treatment, each course of treatment of 4 days. The TCM group were treated with Xiaozhi decoction twice every day for 6 weeks. The IL-1beta, IL-2 and TNF-alpha levels of the peripheral blood and cervical mucous of the two groups were measured by the Ria testing before and after the treatment, and the mycoplasma culture (-) of 32 infertile women as set for control.

RESULTBefore the treatment, TNF-alpha and IL-1beta in levels of the two treatment groups were higher than those of the control group (P < 0.01). In the TCM group, TNF-alpha and IL-1beta levels showed significant differences compared with those before the treatment (P < 0.05) and those of the western group after the treatment (P < 0.01); and IL-2 level didn't have significant change before and after the treatment. The cytokines in peripheral blood of the two treatment groups showed notable difference compared with those of the control group (P < 0.01). In TCM group, IL-2 level had remarkable difference compared with that before the treatment (P < 0.01) and compared with the control group after the treatment (P < 0.01).

CONCLUSIONCytokines (IL-1beta, IL-2, TNF-alpha) in the peripheral blood and cervical mucous increase in infertile women with the mycoplasma infection, suggesting that TCMs can effectively inhibit the levels of IL-1beta, IL-2, TNF-alpha in the peripheral blood and IL-1beta, TNF-alpha in cervical mucous. It is proved that Xiaozhi decoction can be used to treat infertile women with mycoplasma infection.

Adult ; Cytokines ; blood ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Humans ; Infertility, Female ; blood ; complications ; drug therapy ; immunology ; Mycoplasma Infections ; blood ; complications ; drug therapy ; immunology ; Young Adult

The study was aimed to investigate the promotive effect of LRP16 gene on K562 cell proliferation. Open reading frame of LRP16 gene was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and ligated to pGEM-T plasmid to construct LRP16 ORF-pGEM-T recombinant vector. Then, LRP16 ORF identified by sequencing was inserted into pcDNA3.1+ plasmid to construct LRP16 ORF-pcDNA3.1+ recombinant expression plasmid which was transfected into K562 cell lines to make overexpression of LRP16 gene in K562 cells. Survival of cells was determined by MTT assay and growth curve of cells was drawn, the cell cycle was detected by flow cytometry. The results showed that LRP16 ORF was successfully amplified, then the LRP16 ORF-pcDNA3.1+ recombinant plasmid was constructed. The K562 cell line with overexpression of LRP16 gene was established. The promotive effect of LRP16 gene overexpression on proliferation of K562 cells was observed and the effect partially related to the enhancement of cells from G0 to S phase induced by LRP16 gene. It is concluded that LRP16 gene overexpression shows a promotive effect on proliferation of K562 cells.
Cell Proliferation ; Genetic Vectors ; Humans ; K562 Cells ; Neoplasm Proteins ; genetics ; Open Reading Frames ; Plasmids

This study was purposed to investigate lrp16 gene expression in leukemia cell lines and bone marrow cells of leukemia patients and explore the relationship between lrp16 gene expression and development of leukemia. Reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to test the lrp16 mRNA expression in 4 leukemia cell lines, including K562 (CML), HL-60 (APL), MOLT4 (ALL) and U937 cell lines, as well as in bone marrow-derived cells from 115 patients with leukemia. The effect of lrp16 gene expression on genesis and progression of leukemia was analyzed according to clinicopathological features. The results indicated that positive expression of lrp16 mRNA was found in all 4 leukemia cell lines. For leukemia patients, the positive expression rate of lrp16 mRNA in all AML patients was 38% (16/42), in which the positive rates in AML patients with complete remission (CR) and AML patients without remission were 13% (4/30) and 100% (12/12) respectively. The positive expression rate of lrp16 mRNA in ALL patients was 38% (10/26), in which the positive rate in ALL patients with CR and ALL patients without remission were 16% (3/18) and 87% (7/8) respectively. The positive expression rate of lrp16 mRNA in CML patients was 36% (9/25), in which the positive rates in CML patients with CR and CML patients without remission were 20% (4/20) and 100% (5/5) respectively. The positive rate of lrp16 mRNA in CLL patients was 31% (7/22), in which the positive rate in CLL patients with CR and CLL patients without remission were 11% (2/17) and 100% (5/5) respectively. There was no difference of lrp16 gene expression between leukemia subtypes, but there was statistical significant difference in lrp16 gene expression between CR patients and non CR patients (p < 0.001). It is concluded that the lrp16 gene is a leukemic oncogene and closely relates to genesis and progression of leukemia, which may be an indicator for evaluating clinical efficacy of leukemia therapy.
Adolescent ; Adult ; Aged ; Bone Marrow ; metabolism ; pathology ; Female ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; Young Adult

Nowadays, the injury in human mitochondrial DNA (mtDNA) is well known to accumulate in various tissues with age. It's significant to further investigate and then apply it to estimation of the age at parenchymas.
Aging/physiology* ; Base Pair Mismatch/genetics* ; DNA Damage/physiology* ; DNA Fragmentation/genetics* ; DNA, Mitochondrial/physiology* ; Gene Deletion ; Humans ; Polymerase Chain Reaction

To study the methylation in the promoter of LRP15 gene and its relationship with gene expression and to explore the possible mechanism of regulating LRP15 gene methylation, the methylation in the promoter of LRP15 gene in K562 cell line was detected by MS-PCR. Then K562 was exposed to 5-aza-2'-deoxycytidine (CdR) and trichostatin (TSA), to determine whether the silencing of LRP15 gene by de novo methylation could be reversed. As a result, it was confirmed by MS-PCR that the promoter of LRP15 was hypermathylated in K562 cell line, and lost its transcription activity. After CdR, with or without TSA, the silencing of LRP15 gene by de novo methylation can be reversed. Observation demonstrated that the expression of LRP15 was controlled by methylation in its promoter in K562. It is suggested that methyltransferase inhibitor and deacetylase inhibitor may be effective agents in leukemia therapy.
Azacitidine ; analogs & derivatives ; pharmacology ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; K562 Cells ; Neoplasm Proteins ; biosynthesis ; genetics ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; genetics

OBJECTIVETo investigate the methylation frequencies of multiple tumor suppressor genes (TSGs) in hepatocellular carcinoma (HCC) and the clinical implication of aberrant DNA methylation in molecular carcinogenesis of HCC.

METHODSSixty samples of HCC and the paired adjacent liver tissue, 16 samples from post-hepatitis cirrhotic livers, 5 from livers with chronic hepatitis and 5 from normal livers were collected. Eight TSGs frequently silenced by hypermethylation of their promoters in various types of digestive tumors were selected, including APC, RASSF1A, p16, GSTP1, MGMT, DAPK, SOCS-1 and RIZ1. The status of promoter methylation in these 8 genes was investigated using methylation-specific polymerase chain reaction. The clinicopathological data of HCC were also analyzed in order to evaluate the clinical implication of aberrant methylation in HCC.

RESULTSMethylation of the 8 TSGs was quite frequent in HCC, with a methylation rate of 95.0% in RASSF1A, 90.0% in APC, 73.3% in GSTP1, 65.0% in p16, 61.6% in RIZ1 and 60.0% in MGMT. Methylation of the 6 genes was more frequent in HCC than that in adjacent tissues (P < 0.05). The methylation rate of MGMT, GSTP1 and RIZ1 in the adjacent tissues was 41.6%, 40.0% and 25.0%, respectively, significantly higher than that in cirrhotic liver (P < 0.05). p16 methylation was more frequently observed in HCC in elderly patients. The frequency of MGMT methylation was tended to be higher in giant HCC than that in the other types of HCC. Patients with MGMT methylation in the tumor were found to have a shorter disease free survival.

CONCLUSIONDifferent frequency of methylation in hepatocellular carcinomas, adjacent liver tissues and cirrhotic livers implies that epigenetic alteration in the hepatocellular carcinogenesis may be a gradually progressive process. Methylation status of MGMT, GSTP1 and RIZ1 may be promising in risk assessment of hepatocellular carcinoma and in early diagnosis. Furthermore, MGMT methylation might be also used as a potential prognostic biomarker for hepatocellular carcinoma patients.

Adult ; Aged ; Base Sequence ; Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; DNA Modification Methylases ; genetics ; metabolism ; DNA Repair Enzymes ; genetics ; metabolism ; DNA, Neoplasm ; genetics ; DNA-Binding Proteins ; genetics ; metabolism ; Disease-Free Survival ; Female ; Follow-Up Studies ; Gene Expression Regulation, Neoplastic ; Genes, Tumor Suppressor ; Glutathione S-Transferase pi ; genetics ; metabolism ; Hepatitis B, Chronic ; genetics ; metabolism ; Histone-Lysine N-Methyltransferase ; Humans ; Liver ; metabolism ; Liver Cirrhosis ; genetics ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; Male ; Middle Aged ; Molecular Sequence Data ; Neoplasm Recurrence, Local ; Nuclear Proteins ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism ; Young Adult